WO2021170799A1 - Lipases, compositions, procédés et utilisations de celles-ci - Google Patents

Lipases, compositions, procédés et utilisations de celles-ci Download PDF

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Publication number
WO2021170799A1
WO2021170799A1 PCT/EP2021/054825 EP2021054825W WO2021170799A1 WO 2021170799 A1 WO2021170799 A1 WO 2021170799A1 EP 2021054825 W EP2021054825 W EP 2021054825W WO 2021170799 A1 WO2021170799 A1 WO 2021170799A1
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WIPO (PCT)
Prior art keywords
seq
cheese
fatty acids
release
compared
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PCT/EP2021/054825
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English (en)
Inventor
Christian Jaeckel
Hans RAJ
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Chr. Hansen A/S
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Application filed by Chr. Hansen A/S filed Critical Chr. Hansen A/S
Priority to EP21707283.4A priority Critical patent/EP4110907A1/fr
Priority to AU2021227090A priority patent/AU2021227090A1/en
Priority to CA3163711A priority patent/CA3163711A1/fr
Priority to US17/798,153 priority patent/US20230098388A1/en
Priority to JP2022551279A priority patent/JP2023515570A/ja
Publication of WO2021170799A1 publication Critical patent/WO2021170799A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/02—Making cheese curd
    • A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
    • A23C19/0328—Enzymes other than milk clotting enzymes, e.g. lipase, beta-galactosidase
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C13/00—Cream; Cream preparations; Making thereof
    • A23C13/12—Cream preparations
    • A23C13/16—Cream preparations containing, or treated with, microorganisms, enzymes, or antibiotics; Sour cream
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C15/00—Butter; Butter preparations; Making thereof
    • A23C15/12—Butter preparations
    • A23C15/123—Addition of microorganisms or cultured milk products; Addition of enzymes; Addition of starter cultures other than destillates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C20/00—Cheese substitutes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
    • A23C9/1216—Other enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)

Definitions

  • the present invention relates to a wild-type, non-engineered, microbial lipolytic enzyme with a high specificity for short-chain fatty acids, preferably with a higher specificity for short chain fatty acids than for medium to long chain fatty acids, preferably when compared to a known microbial lipolytic enzyme, a nucleic acid encoding said lipolytic enzyme, a vector, a host cell and/or a composition comprising the lipolytic enzyme.
  • This invention also relates to a process for preparing a food product, such as a dairy product, wherein the lipolytic enzyme is used.
  • This invention is suitable for use in the food industry, preferably in the dairy industry, more preferably in the cheese industry leading, for example, to flavor enhancement or shortening of the ripening times for ripened cheeses.
  • Lipolytic enzyme also called lipases (EC 3.1.1.3) are a class of hydrolases that hydrolyze ester bonds of lipids of triglycerides generating free fatty acids. This class of enzymes is responsible, for example, for the lipolysis of milk fat and/or other fat and subsequent release of short (C4- to C 6 -fatty acids), medium and/or long (Cs- and higher) chain fatty acids.
  • Each lipolytic enzyme has its own release profile of free fatty acids from milk fat and/or other fat. This profile may range from short (C4- to C 6 -fatty acids), to medium and/or long (Cs- and higher) chain fatty acids.
  • the mixture of short (C4- to C 6 -fatty acids), medium and/or long (Cs- and higher) chain fatty acids obtained is dependent, at least, on the lipolytic enzyme and on the lipolytic substrate.
  • the mixture of short (C4- to C 6 -fatty acids), medium and/or long (Cs- and higher) chain fatty acids obtained is responsible for modulating the flavor of a given final product.
  • a mixture having a higher amount or concentration of short chain fatty acids such as a higher amount or concentration of butyric acid (C4-fatty acid)
  • an amount or concentration of medium and/or long chain fatty acids leads to the development of a preferred rancid cheese flavor, in particular during ripening, and has been used for flavor generation in dairy products, for example in various cheese types.
  • a mixture having a higher amount or concentration of medium and/or long chain fatty acids, specially a mixture having a higher amount or concentration of long chain fatty acids (C16- and higher), than an amount or concentration of short chain fatty acids gives rise to off flavors.
  • Lipolytic enzymes or lipases can be of animal origin or non-animal origin.
  • Lipases from animal origin preferably cleave short chain fatty acids (C4- to C 6 -fatty acids) from milk fat and/or other fat rather than long chain fatty acids.
  • These short chain fatty acids are responsible for the formation of the preferred rancid taste rather than the soapy taste produced by the medium to long chain fatty acids.
  • C ⁇ fatty acids are responsible for the formation of the preferred rancid taste rather than the soapy taste produced by the medium to long chain fatty acids.
  • medium chain fatty acids which add animal specific taste to the cheese.
  • lipases from animal origin cannot be used in vegetarian and/or kosher products, which is a highly demanded market.
  • lipases from non-animal origin such as from microbial origin
  • microbial lipases are known for being non-specific lipases mainly releasing medium to long chain fatty acids (Cs- and higher) from milk fat and/or other fat, thereby giving cheese an extremely unpleasant soapy taste.
  • Microbial lipases can be obtained, for example, from Aspergillus, Rhizopus, and Mucor.
  • the effect of these fungal lipases for developing rancidity in fats with respect to Aspergillus is disclosed by Iwai et al. in J. Gen. Appl. Microbiol. 10, 13-22 (1964), US 4 726 954 or US 4 636 468; with respect to Rhizopus is described by Oi et al. in Agr. Biol. Chem. 33, 729-38 (1969); and with respect to Mucor is set forth by Somkuti et al. in Appl. Microbiol. 16, 617- 19 (1968).
  • lipases have a higher specificity towards medium to long chain fatty acids, such as Aspergillus or Rhizopus, or towards medium chain fatty acids, as it is the case of Mucor, than towards short chain fatty acids, such as C4- to C6-fatty acids.
  • Microbial lipases can also be obtained from Yarrowia, such as Yarrowia (Y.) lipolytica.
  • Yarrowia Yarrowia
  • Several documents disclose the preference of lipases from Yarrowia towards medium to long chain fatty acids.
  • Kamoun et al. 2015 describes a higher specificity for trioctanoin than for tributyrin at pH 7.5 for Lip8;
  • Aloulou et al. 2007 discloses a higher specificity for trioctanoin than for tributyrin at pH 6 for Lip2; Sheng, J., et al. 2012 mentions the LipY has the highest activity on Cs-Ci2 fatty acids.
  • US2005059130 also describes microbial lipases derived from Humicola lanuginosa, Fusarium oxysporum or Rhizomucor miehei (examples 10, 11 or 12, respectively), wherein the substrate specificity of the wild type enzymes was modified by making alterations to the amino acid sequences of said lipases and as such obtain variants with increased specificity for short-chain fatty acids.
  • EP 3 081 644 discloses a modified lipase from Candida cylindracea also having higher specificity towards short-chain fatty acids.
  • none of these microbial engineered lipases is a naturally occurring lipase with a higher specificity for short chain fatty acids than for medium to long chain fatty acids.
  • the foregoing illustrates the difficulty in obtaining a microbial wild-type lipase with higher specificity towards short chain fatty acids, specially towards C ⁇ fatty acids, and a lower specificity towards medium and/or long chain fatty acids, specially a lower specificity towards long chain fatty acids, leading to a reduction in soapiness and an increase in butyric flavors in a food product, such as a dairy product or cheese, and therefore able to replace the microbial lipases currently available.
  • the present invention provides a novel wild-type, microbial lipolytic enzyme suitable to be used in the food industry, preferably in the dairy industry, more preferably in the cheese industry.
  • the enzymes herein disclosed have a higher specificity towards short chain fatty acids, in particular C ⁇ fatty acids and/or C 6 -fatty acids, when compared to a known microbial lipolytic enzyme under the same conditions, in particular for the same dosage of enzyme and in the same fat matrix, meaning that these enzyme release or produce or generate more short chain fatty acids than known microbial enzyme(s).
  • wild-type microbial lipolytic enzymes were known to be specific towards medium or long chain fatty acids and not short chain fatty acids, preferably not specific towards C ⁇ fatty acids and/or C 6 -fatty acids.
  • This invention relates to a lipase comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or
  • SEQ ID NO: 10 or SEQ ID NO: 11 preferably SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO:
  • SEQ ID NO: 9 SEQ ID NO: 11, more preferably SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 11, even more preferably SEQ ID NO: 1.
  • This invention also relates to a lipase comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11, preferably SEQ ID NO: 1 or SEQ ID NO:
  • This invention also relates to a lipase comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11, preferably SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 9 or SEQ ID NO: 11, more preferably SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 11, even more preferably SEQ ID NO: 1, or having at least 95%, 96%, 97%, 98% or 99% identity SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11, preferably SEQ ID
  • the lipase herein disclosed - SEQ ID NO: 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 - is an isolated microbial lipase, a recombinant microbial lipase or a synthetic microbial lipase.
  • the present invention relates to a wild-type, non-engineered, microbial lipolytic enzyme
  • one or more alterations may be made to any of the amino acid sequence of the enzymes herein disclosed without interfering with the activity of the said enzyme and preferably without changing the C4 : o fatty acid preference of the sequence.
  • Such alteration can be an addition, a substitution or a deletion.
  • a change in the N-terminal or C-terminal of any one of the sequences herein disclose may lead to a different sequence without, however, changing the fatty acid preference of the changed sequence versus the unchanged one.
  • This invention also relates to an isolated DNA sequence or recombinant DNA sequence or synthetic DNA sequence, wherein the isolated DNA sequence or recombinant DNA sequence or synthetic DNA is selected from a sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98% or at least 99%, or 100% sequence identity with SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 18 or SEQ ID NO: 19 or SEQ ID NO: 20 or SEQ ID NO: 21 or SEQ ID NO: 22 or SEQ ID NO: 23.
  • said DNA sequence may further comprise a signal peptide sequence.
  • This invention further concerns a vector, such as a cloning vector and/or an expression vector, comprising an isolated DNA sequence or recombinant DNA sequence or synthetic DNA sequence encoding the lipase SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO:
  • the DNA sequence may be SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15 or
  • the sequence is cloned into the vector using standard cloning techniques. The selection of the cloning vector and/or expression vector is dependent on the host cell, as the cloning vector and/or expression vector need to be compatible with the host cell.
  • the invention also relates to a host cell, preferably a recombinant host cell, comprising SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO:
  • the invention also concerns a method of growing, transforming or transfecting said vector in a suitable host cell and in a condition leading to expression of the polypeptide or to the over expression of the polypeptide.
  • a suitable host cell used in this invention may be any host cell in which a recombinant DNA can be replicated and a gene encoding an enzyme can be expressed.
  • the host cell may be a mammalian cell, an insect cell, a microbial cell, e.g. a bacterial cell or a fungal cell wherein a yeast cell is included.
  • yeasts may be Saccharomyces spp., Schizosaccharomyces spp., Candida spp., Pichia spp., Kluyveromyces spp., such as Saccharomyces cerevisiae ; Candida cylindracea, or Pichia pastoris.
  • suitable fungal cells including filamentous fungal cells
  • suitable bacteria are Gram-negative such as Escherichia coli or Gram-positive bacteria selected from Bacillus spp., Streptomyces spp., Corynebacterium spp., such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, ⁇ Streptomyces lividans or Streptomyces murinus.
  • the transformation of bacteria may, for instance, be effected by using competent cells in a manner known in the art or by any other way known in the art.
  • the host cell is selected from Pichia pastoris, Saccharomyces spp., Saccharomyces cerevisiae, Schizosaccharomyces spp., Candida spp., Candida cylindracea, Kluyveromyces spp., Fusarium spp., Fusarium oxysporium, Aspergillus spp., Aspergillus oryzae or Aspergillus niger, Trichoderma spp., Escherichia coli or Bacillus spp., Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, Streptomyces spp., Str
  • the invention also relates to a lipase consisting of an amino acid sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11, preferably having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 9 or SEQ ID NO: 11; wherein the lipase is expressed in Pichia pastoris and has a higher specificity towards the release of C ⁇ fatty acids from a dairy composition comprising milk fat and/or other fat if
  • the present invention also concerns a method for preparing a food product
  • - comprising a step of using or adding a lipase comprising an amino acid sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11, preferably having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 9 or SEQ ID NO: 11, preferably, wherein the food product has a higher content or amount or concentration of short chain fatty acids, such as C4- and/or C6-fatty acids, than if compared
  • - comprising a step of using or adding a lipase encoded by a DNA sequence selected from a sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98% or at least 99%, or 100% sequence identity with SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 18 or SEQ ID NO: 19 or SEQ ID NO: 20 or SEQ ID NO: 21 or SEQ ID NO: 22 or SEQ ID NO: 23, preferably having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98% or at least 99%, or 100% sequence identity with SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 20 or SEQ ID NO: 22 or SEQ ID NO: 23, preferably wherein the food product has a higher content or amount or concentration of short chain fatty
  • the food product may be a dairy product, as a cheese product, a processed cheese, a cheese-like product, or an enzyme-modified cheese, or a butter, a yogurt, a cream, a seasoning.
  • the dairy product is a cheese product, preferably wherein the cheese product is Pecorino, Provolone, Parmesan, Grana Padano, Pgiano Reggiano, Romano, Chester, Danbo, Manchego, Saint Paulin, Cheddar, Monterey, Colby, Edam, Gouda, Muenster, Swiss type, Gruyere, Emmental; curd-cheeses such as Feta cheese; pasta filata cheeses such as Mozzarella, and Queso fresco cheese; fresh cheese such as Ricotta, Cream cheese, Neufchatel or Cottage cheese; cream cheese, white mold cheese such as Brie and Camembert cheese, blue mold cheese such as Gorgonzola and Danish blue cheese; and processed cheese, preferably Feta cheese or Provolone cheese.
  • the cheese product is Pecorino, Provolone, Parmesan, Grana Padano, Peauo Reggiano, Romano, Chester, Danbo, Manchego, Saint Paulin, Cheddar, Monterey, Colby, Ed
  • the food product may be a non-dairy product, preferably a non-dairy cheese or vegan cheese, more preferably wherein the lipase has a higher specificity towards the release of C ⁇ fatty acids from a composition comprising fat such as vegetable fat or from a water-in-oil emulsion if compared with the release of Cio-fatty acids or if compared with the release of Cs-fatty acids, or if compared with the release of C 6 -fatty acids, or if compared with the release of Ci2-fatty acids or if compared with the release of Cis : 2-fatty acids or if compared with the release of Ci4-fatty acids or if compared with the release of Cis : o-fatty acids or if compared with the release of Cis :i -fatty acids or if compared with the release of Ci 6 -fatty acids, or if compared with the release of Cs-fatty acids - Cis : 2-fatty acids, or if compared with the release of Cio
  • cheese flavor or cheese intensity can be increased and/or improved by applying a lipase of the present invention.
  • the increase or improvement of cheese flavor or cheese intensity can apply to cheese itself or to processed cheese, to cheese like product or to enzyme-modified cheese or even to non-dairy cheese such as vegan cheese.
  • the lipases herein disclosed may be added, for example, to an intermediate product during the process of producing a food product or to a raw material such as milk, in particular milk obtained from a cow, sheep, goat, among others.
  • the invention also concerns the use of a lipase comprising an amino acid sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11, preferably having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 9 or SEQ ID NO: 11, for producing a food product or as an agent for producing a food product, preferably wherein the food product has a higher content or amount or concentration of short chain fatty acids, such as C4- and/
  • a lipase is for producing a food product, in particular a dairy product or a cheese such Pecorino, Provolone, Parmesan, Grana Padano, Pgiano Reggiano, Romano, Chester, Danbo, Manchego, Saint Paulin, Cheddar, Monterey, Colby, Edam, Gouda, Muenster, Swiss type, Gruyere, Emmental; curd-cheeses such as Feta cheese; pasta filata cheeses such as Mozzarella, and Queso fresco cheese; fresh cheese such as Ricotta, Cream cheese, Neufchatel or Cottage cheese; cream cheese, white mold cheese such as Brie and Camembert cheese, blue mold cheese such as Gorgonzola and Danish blue cheese, preferably Feta cheese or Provolone cheese; or processed cheese.
  • a dairy product or a cheese such Pecorino, Provolone, Parmesan, Grana Padano, Peauo Reggiano, Romano, Chester, Danbo, Manchego, Saint Paulin
  • the invention also relates to the use of a lipase for producing or releasing more C ⁇ fatty acids from a dairy composition comprising milk fat and/or other fat, than for producing or releasing more C 6 -fatty acids, Cs-fatty acids, Cio-fatty acids, Ci2-fatty acids, Ci4-fatty acids, Ci 6 -fatty acids, Ci 8: o-fatty acids, Cis :i -fatty acids, or Cis : 2-fatty acids, or Cis : 3-fatty acids, wherein the lipase comprises an amino acid sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11, preferably having at least 90%,
  • the invention also concerns the use of a lipase for producing or releasing more C4-fatty acids and/or C 6 -fatty acids in a food product than for producing or releasing, Cs-fatty acids, C10- fatty acids, Ci2-fatty acids, Ci4-fatty acids, Ci 6 -fatty acids, Cis : o-fatty acids, Cis :i -fatty acids, or Cis : 2-fatty acids, or Cis : 3-fatty acids in the food product, wherein the lipase comprises an amino acid sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11, preferably having at least 90%, or at least
  • the food product is a dairy product selected from a cheese and wherein the cheese is Feta cheese, or Provolone cheese, or Pecorino cheese, or Parmesan cheese, or Grana Padano cheese, or Policyo Reggiano cheese, or Romano cheese, or Chester cheese, or Danbo cheese, or Manchego cheese, or Saint Paulin cheese, or Cheddar cheese, or Monterey cheese, or Colby cheese, or Edam cheese, or Gouda cheese, or Muenster cheese, or Swiss type cheese, or Gruyere cheese, or Emmental cheese; or pasta filata cheeses such as Mozzarella, and Queso fresco cheese; or fresh cheese such as Ricotta, Cream cheese, Neufchatel or Cottage cheese; or cream cheese, or white mold cheese such as Brie and Camembert cheese, or blue mold cheese such as Gorgonzola and Danish blue cheese, preferably Feta cheese or Provolone cheese.
  • the cheese is Feta cheese, or Provolone cheese, or Pecorino cheese, or Parmesan cheese,
  • the invention concerns a food product obtainable by using a lipase SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11, preferably wherein the food product is a dairy product such as a cheese product, a processed cheese, a cheese-like product, or a butter, a yogurt, a cream, a seasoning, more preferably wherein the dairy product is a cheese, such Pecorino, Provolone, Parmesan, Grana Padano, Personalgiano Reggiano, Romano, Chester, Danbo, Manchego, Saint Paulin, Cheddar, Monterey, Colby, Edam, Gouda, Muenster, Swiss type, Gruyere, Emmental; curd-cheeses such as Feta cheese; pasta filata cheeses such as
  • the invention is related as well with a food product comprising a lipase, wherein the lipase comprises an amino acid sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11, preferably having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 9 or SEQ ID NO: 11, and wherein the food product comprises more C ⁇ fatty acids than Cio-fatty acids or than any other fatty acid selected from C 6 -fatty acids or
  • the food product is a dairy product selected from a cheese, a processed cheese, a cheese-like product, an enzyme-modified cheese, or a butter, a yogurt, a cream, a seasoning, preferably wherein the cheese is Feta cheese, or Provolone cheese, or Pecorino cheese, or Parmesan cheese, or Grana Padano cheese, or Policyo Reggiano cheese, or Romano cheese, or Chester cheese, or Danbo cheese, or Manchego cheese, or Saint Paulin cheese, or Cheddar cheese, or Monterey cheese, or Colby cheese, or Edam cheese, or Gouda cheese, or Muenster cheese, or Swiss type cheese, or Gruyere cheese, or Emmental cheese; or pasta filata cheeses such as Mozzarella, and Queso fresco cheese; or fresh cheese such as Ricotta, Cream cheese, Neufchatel or Cottage cheese; or cream cheese, or white mold cheese such as Brie and Camembert cheese, or blue mold cheese such as Gorgonzola and Danish blue
  • the food product is a non-dairy product such as a non-dairy cheese or vegan cheese.
  • the invention also relates to a composition
  • a composition comprising a lipase according to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11, and/or a DNA sequence according SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 18 or SEQ ID NO: 19 or SEQ ID NO: 20 or SEQ ID NO: 21 or SEQ ID NO: 22 or SEQ ID NO: 23, and/or a vector comprising any of the sequences SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or S
  • short chain fatty acid means “short chain fatty acid”, “medium chain fatty acid”, “long chain fatty acid”, “FFA”, “lipase”, “lipolytic enzyme”, “microbial lipase”, “microbial lipolytic enzyme”, “lipase with higher specificity towards the release of short-chain fatty acids”, “lipase with higher specificity towards the release of C ⁇ fatty acids”, “lipase with higher specificity towards the release of C 6 -fatty acids”, lipase with higher specificity towards the release of medium and/or long-chain fatty acids”, “lipase activity”, “lipase dosage”, “wild type”, “synthetic sequence”, “isolated sequence”, “recombinant sequence”, “mature protein sequence”, “expression vector”, “sequence identity”, “food product”, “dairy product”, “cheese”, “cheese product”, “processed cheese”, “enzyme-modified cheese”, “cheese-like product”, “improvement factor (IF)
  • short chain fatty acid means a fatty acid comprising 4-6 carbons.
  • Examples of short chain fatty acids are: butyric acid (butanoic acid; C 4 ), valeric acid (pentanoic acid; C 5 ) or caproic acid (hexanoic acid; Ce) ⁇
  • medium chain fatty acid means a fatty acid comprising 8-12 carbons.
  • medium chain fatty acids are: caprylic acid (octanoic acid; Cs), pelargonic acid (nonanoic acid; C 9 ), capric acid (decanoic acid; C 10 ), undecylic acid (undecanoic acid; Cn) or lauric acid (dodecanoic acid; C 12 ).
  • long chain fatty acid means a fatty acid comprising 14-18 or more carbons.
  • long chain fatty acids are: myristic acid (tetradecanoic acid; C 14 ), pentadecanoic acid (C 15 ), palmitic acid (hexadecanoic acid; Ci 6 ), margaric acid (heptadecanoic acid; C 17 ), stearic acid (octadecanoic acid; Cis).
  • FFA free fatty acid
  • lipase and “lipolytic enzyme” are used interchangeably.
  • microbial lipase and "microbial lipolytic enzyme” are used interchangeably, and as used herein means a lipase or a lipolytic enzyme expressed by a naturally occurring microorganism found in nature.
  • the microorganism may be a filamentous fungus and/or a non-filamentous fungus.
  • the lipase or the microbial lipase or the microbial lipolytic enzyme may be a filamentous fungus lipase and/or non-filamentous fungus lipase.
  • lipase with higher specificity towards the release of short-chain fatty acids from a dairy composition comprising milk fat and/or other fat, means that said lipase preferably hydrolyzes ester bonds of lipids of triglycerides generating more C ⁇ fatty acids and/or more C6-fatty acids rather than generating medium and/or long chain fatty acids.
  • lipase with higher specificity towards the release of C ⁇ fatty acids from a dairy composition comprising milk fat and/or other fat, means that said lipase preferably hydrolyzes ester bonds of lipids of triglycerides generating more C ⁇ fatty acids rather than generating other kinds of fatty acids, such as medium and/or long chain fatty acids.
  • lipase with higher specificity towards the release of C ⁇ fatty acids from a dairy composition comprising milk fat and/or other fat, if compared with the release of Cio-fatty acids means that said lipase preferably hydrolyses ester bonds of lipids of triglycerides generating more C 4 -fatty acids rather than generating Cio-fatty acids.
  • the term "lipase with higher specificity towards the release of C ⁇ fatty acids", from a dairy composition comprising milk fat and/or other fat, if compared with the release of Cs-fatty acids or C6-fatty acids or Ci 2 -fatty acids or Cis :2 -fatty acids or Ci 4 -fatty acids or Cis : o-fatty acids or Cis :i -fatty acids or Ci6-fatty acids means that said lipase preferably hydrolyses ester bonds of lipids of triglycerides generating more C 4 -fatty acids rather than generating Cs-fatty acids or C6-fatty acids or Ci 2 -fatty acids or Cis :2 -fatty acids or Ci 4 -fatty acids or Cis : o-fatty acids or Cis :i -fatty acids or Ci6-fatty acids, respectively.
  • the molar fraction of C 4 -fatty acids is higher than the molar fraction of medium and/or long chain fatty acids, for the same tested conditions, preferably the molar fraction of C 4 -fatty acids is higher than the molar fraction of other fatty acids, such as C6-fatty acids or Cs-fatty acids or Cio-fatty acids or Ci 2 -fatty acids or Ci 4 -fatty acids or Ci6-fatty acids or Cis : o-fatty acids or Cis :i -fatty acids or Cis :2 -fatty acids.
  • lipase with higher specificity towards the release of C 4 -fatty acids from a dairy composition comprising milk fat and/or other fat, also means that said lipase preferably hydrolyzes ester bonds of lipids of triglycerides generating more C 4 -fatty acids than a control microbial lipase.
  • the molar fraction of C 4 -fatty acids of the tested lipase is higher than the molar fraction of C 4 -fatty acids of the control microbial lipase.
  • lipase with higher specificity towards the release of C6-fatty acids from a dairy composition comprising milk fat and/or other fat, means that said lipase preferably hydrolyzes ester bonds of lipids of triglycerides generating more C6-fatty acids rather than generating other kinds of fatty acids, such as medium and/or long chain fatty acids.
  • lipase with higher specificity towards the release of C6-fatty acids from a dairy composition comprising milk fat and/or other fat, if compared with the release of Cio-fatty acids means that said lipase preferably hydrolyses ester bonds of lipids of triglycerides generating more C6-fatty acids rather than generating Cio-fatty acids.
  • the term "lipase with higher specificity towards the release of C6-fatty acids", from a dairy composition comprising milk fat and/or other fat, if compared with the release of Cs-fatty acids or Ci 2 -fatty acids or Cis : 2-fatty acids or Ci4-fatty acids or Cis : o-fatty acids or Cis :i -fatty acids or Ci 6 -fatty acids means that said lipase preferably hydrolyses ester bonds of lipids of triglycerides generating more C 6 -fatty acids rather than generating Cs-fatty acids or Ci2-fatty acids or Cis : 2-fatty acids or Ci4-fatty acids or Cis : o-fatty acids or Cis :i -fatty acids or Ci 6 -fatty acids, respectively.
  • the molar fraction of C 6 -fatty acids is higher than the molar fraction of medium and/or long chain fatty acids, for the same tested conditions, preferably the molar fraction of C 6 -fatty acids is higher than the molar fraction of other fatty acids, such as Cs-fatty acids or Cio-fatty acids or Ci2-fatty acids or Ci4-fatty acids or Ci 6 -fatty acids or Cis : o-fatty acids or Cis :i -fatty acids or Cis : 2-fatty acids.
  • lipase with higher specificity towards the release of C 6 -fatty acids from a dairy composition comprising milk fat and/or other fat, also means that said lipase preferably hydrolyzes ester bonds of lipids of triglycerides generating more C 6 -fatty acids than a control microbial lipase.
  • the molar fraction of C 6 -fatty acids of the tested lipase is higher than the molar fraction of C4-fatty acids of the control microbial lipase.
  • lipase with higher specificity towards the release of medium and/or long-chain fatty acids from a dairy composition comprising milk fat and/or other fat, means that said lipase preferably hydrolyzes ester bonds of lipids of triglycerides generating Cs- to Cis-fatty acids rather than generating C4-fatty acids and/or C 6 -fatty acids.
  • the molar fraction of Cs- to Cis-fatty acids of the tested lipase is higher than the molar fraction of Cs- to Cis- fatty of the control microbial lipase.
  • C4-fatty acids and “C4 : o-fatty acids”; “C 6 -fatty acids” and “C 6: o-fatty acids”; “Cs-fatty acids” and “Cs : o- fatty acids”; “Cio-fatty acids” and “Cio : o-fatty acids”; “Ci2-fatty acids” and “Ci2 : o-fatty acids”; “Ci4-fatty acids” and “Ci4 : o-fatty acids”.
  • the term”Ci 6 -fatty acids” may include Ci 6: o-fatty acids and Ci 6:i -fatty acids”.
  • the term “Cis-fatty acids” may include Cis : o-fatty, Cis :i -fatty acids and Cis :3 -fatty.
  • lipase activity and “lipase dosage” are used interchangeably.
  • the "lipase activity” may be determined, for dosing in cheese and cream, for example by the International Dairy Federation (IDF) method for both the commercial lipase and the enzymes herein disclosed (SEQ ID NO 1-11).
  • the IDF method mainly, corresponds to the International Standard ISO 13082:2011, 1 st edition of 2011-11-15, IDF 218:2011 (also labelled as ISO 13082
  • the term “LFU/L of milk” is used.
  • LFU/L of milk relates to the concentration or amount of enzyme or dosage of enzyme per liter of milk used to produce a dairy product, such as cheese.
  • LFU/L of milk means lipase forestomach units per liter of milk.
  • One LFU is defined as the amount of lipase activity that releases butyric acid at a rate of 1.25 pmol/min, under the tested conditions.
  • the lipase activity may also be determined as explained in the Examples below.
  • there are other methods of determining lipase activity which has been disclosed in prior art documents (such as in EP 3 081 644, EP 2 254 996 or EP 1 776 455). However, what is relevant is that within the same example or within comparative examples, the lipase activity is determined in the same way for all lipases under analysis, including the control lipase.
  • wild type lipase means, in the context of the present invention, a lipase whose sequence has not been mutated, i.e. does not contain amino acid deletions, additions or substitutions, when compared with a mature protein sequence (after co- and/or post- translational cleavage events) endogenously produced.
  • Wild type lipases of the present invention may be encoded by codon optimized polynucleotides for heterologous expression and may also comprise a non-endogenous signal- and/or propeptide selected for expression in a given host.
  • a lipase may also be synthetically obtained or genetically modified, using methods well known in the art, as to reproduce the wild-type lipase.
  • synthetic sequence or a variation thereof means a sequence that has been prepared by in vitro chemical or enzymatic synthesis. It includes, but is not limited to, sequences made with optimal codon usage for host organisms. The term applies to a synthetic microbial lipase or to a synthetic DNA sequence.
  • isolated sequence or a variation thereof means a sequence which is at least substantially free from at least one other component with which the sequence is naturally associated in nature and as found in nature.
  • an "isolated sequence” can also mean a sequence removed from its native environment.
  • sequences which have been recombinantly produced in a host organism are considered isolated for the purpose of this invention as are native or recombinant sequences which have been substantially purified by any suitable technique as such, for example, a purification method. Purification methods are well known in the art. The term applies to an isolated microbial lipase or to an isolated DNA sequence.
  • recombinant sequence or a variation thereof means a sequence that has been prepared using recombinant DNA techniques, which are within the capabilities of a person skilled in the art and are explained in literature, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory manual, 2 nd edition, books 1-3, Cold Spring Harbor Laboratory Press. The term applies to a recombinant microbial lipase or to a recombinant DNA sequence.
  • mature protein sequence used herein means a sequence having lipolytic activity that is in its final form following translation and any co- and/or post-translational cleavage events or modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, among other events or modifications.
  • expression vector means a construct capable of in vivo or in vitro expression.
  • the expression vector may be incorporated, in particular stably incorporated, into the genome of a suitable host organism.
  • sequence identity describes a quantitative measure of similarity between two amino acid sequences or two nucleotide sequences.
  • degree of sequence identity between two amino acid sequences is based on aligning both sequences with the blastp suite provided by the National center for Biotechnology Information (NCBI) on https://blast.ncbi.nlm.nih.gov applying standard parameter settings (Matrix: BLOSUM62, Gap Costs: Existence: 11 Extension :1, Conditional compositional score matrix adjustment) and subsequent quantification of identical amino acid pairs in identical positions over the aligned amino acid sequences.
  • food product refers to a kind of milk-based product intended to be used as food, including, but not limited to, cheese, milk, skimmed milk, acidified milk, butter milk, condensed milk, spread, margarine, yoghurt, ice cream, milk powder, butter, dulce de leche, among others.
  • dairy product is intended to include any food product made using milk or milk- based product, including, but not limited to, cheese, milk, skimmed milk, acidified milk, butter milk, condensed milk, spread, margarine, yoghurt, ice cream, milk powder, butter, dulce de leche, among others.
  • the term “cheese” or “cheese product” are used interchangeably.
  • the term “cheese” is understood to encompass any cheese, including, but not limited to, hard cheeses such as Pecorino, Provolone, Parmesan, Grana Padano, Reggiano Reggiano, Romano, Chester, Danbo, Manchego, Saint Paulin, Cheddar, Monterey, Colby, Edam, Gouda, Muenster, Swiss type, Gruyere, Emmental; curd-cheeses such as Feta cheese; pasta filata cheeses such as Mozzarella, and Queso fresco cheese; fresh cheese such as Ricotta, Cream cheese, Neufchatel or Cottage cheese; cream cheese, white mold cheese such as Brie and Camembert cheese, blue mold cheese such as Gorgonzola and Danish blue cheese; and processed cheese, enzyme-modified cheese (EMC) or cheese-like product.
  • hard cheeses such as Pecorino, Provolone, Parmesan, Grana Padano
  • processed cheese is preferably manufactured from cheese or cheese analogues by cooking and emulsifying the cheese, such as with emulsifying salts (e.g. phosphates and citrate).
  • emulsifying salts e.g. phosphates and citrate.
  • the process may further include the addition of spices/condiments.
  • EMC enzyme-modified cheese
  • cheese curd which has been treated with enzymes to produce a concentrated cheese flavor ingredient which may have approximately 15-30 times the flavor intensity of natural cheese.
  • EMCs are available as pastes or dried to form powders and are used to give a cheese flavor note to products such as processed cheese or analogue cheese, cheese powders, soups, sauces, dips, crackers, salad dressings and in coatings for snack foods.
  • chee-like product is understood as cheese-like products which contain fat, such as e.g. milk fat (e.g. cream or butter) or vegetable oil, as a part of the composition, and which further contain, as part of the composition, one or more non-milk constituents, such as e.g. a vegetable constituent (e.g. vegetable protein or vegetable oil).
  • a "cheese-like product” includes a non-dairy cheese, also known as vegan cheese, wherein the fat used to make the non-dairy cheese is vegetable oil or an emulsion such as water-in-oil emulsion, instead of milk fat.
  • improvement factor is defined as the ratio obtained when dividing the enzymatic specificity for the release of short chain fatty acids of a tested lipase by the enzymatic specificity for the release of short chain fatty acids of a control lipase, preferably a control microbial lipase or is defined as the ratio obtained when dividing the enzymatic specificity for the release of medium chain fatty acids of a tested lipase by the enzymatic specificity for the release of medium chain fatty acids of a control lipase, preferably a control microbial lipase. If the ratio is 1, then the tested lipase has an identical preference, or specificity, for short chain fatty acids as control sequence.
  • the tested lipase has less preference, or specificity, for short chain fatty acids than the control lipase, preferably a control microbial lipase. If the ratio is higher than one, then the tested lipase has higher preference, or specificity, for short chain fatty acids than the control.
  • the enzymatic specificity for short chain fatty acids is obtained by dividing the enzymatic activity for short chain fatty acids C4 or C6 by the enzymatic activity for long chain fatty acids C16-C18.
  • the improvement factor (IF) may also be determined by the ratio obtained when dividing the enzymatic specificity for the release of long chain fatty acids of a tested lipase by the enzymatic specificity for the release of long chain fatty acids of a control lipase, preferably a control microbial lipase. If the ratio is 1, then the tested lipase has an identical preference, or specificity, for longer chain fatty acids as the control lipase, preferably a control microbial lipase. If the ratio is lower than one, then the tested lipase has less preference, or specificity, for long chain fatty acids than the control.
  • the tested lipase has higher preference, or specificity, for long chain fatty acids than the control.
  • the enzymatic specificity for long chain fatty acids is obtained by dividing the enzymatic activity for long chain fatty acids C16-C18 by the enzymatic activity for short chain fatty acids C4 or C 6 -
  • control lipase or "control microbial lipase” or “control lipolytic enzyme” or “control microbial lipolytic enzyme” is a commercial microbial lipase from Rhizomucur miehei (Palatase®, Palatase® 20000 L, available from Novozymes A/S), and herein labelled as commercial microbial lipase or commercial microbial lipase A.
  • Rhizomucur miehei Palatase®, Palatase® 20000 L, available from Novozymes A/S
  • other microbial lipases from Rhizomucur miehei can be used, for example SpicelT ® MR available from Chr. Hansen.
  • signal peptide relates to an amino acid sequence fused to the N-terminus of the polypeptide chain of the enzyme that is recognized by the secretory pathway of the enzyme producing microbial organism to translocate the synthesized protein into the cellular membrane or secrete it out of the cell into the fermentation medium.
  • propeptide relates to a polypeptide chain fused to the N-terminus of the major peptide chain of an enzyme that may support folding of the synthesized protein and/or contribute to its stability.
  • the propeptide is cleaved during maturation or activation of the enzyme.
  • a propeptide acts as an N-terminal extension of protein, distinct from the signal sequence which is necessary for the transport of the protein into or through the membrane, or for its secretion into the extracellular medium.
  • the presence of a propeptide sequence may lead to a stabilization of the expression of lipases and/or may prevent inactivation in vivo of the lipase.
  • Amino acids are herein indicated by their one letter code or by their three letter code, both of which are well known to a person skilled in the art.
  • SEQ ID NO: 1-11 stands for SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.
  • SEQ ID NO: 12-23 stands for SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23.
  • the present invention relates to a non-animal, wild-type derived from a microorganism lipase fulfilling the vegetarian and/or kosher requirements while simultaneously presenting a specificity and preference for the cleavage of short chain fatty acids, preferably presenting a higher specificity towards C ⁇ fatty acids and/or C 6 -fatty acids rather other kinds of fatty acids or presenting a higher specificity towards C ⁇ fatty acids and/or C 6 -fatty acids than a commercial microbial lipase.
  • the purpose of this invention is to provide a microbial wild-type lipase with a higher specificity towards the release of C 4 - fatty acids and/or C 6 -fatty acids, from a dairy composition comprising milk fat and/or other fat, if compared with the release of medium and/or long chain fatty acids, preferably if compared with the release of long-chain fatty acids, or a higher specificity towards the release of C ⁇ fatty acids and/or C 6 -fatty acids, from a dairy composition comprising milk fat and/or other fat, if compared to the release of C ⁇ fatty acids and/or Oe- fatty acids of a commercial microbial lipase, without the need to generate any mutation (addition, deletion and/or substitution) in the lipase sequence.
  • this invention relates to an improved (or alternative) microbial wild-type lipase with specificity towards short-chain fatty acids versus the prior microbial lipases. Therefore, the lipases herein disclosed may be obtained from microorganisms that produce the lipase naturally. In this case, said lipase is a wild-type lipase.
  • the microbial lipases wherein disclosed may be used to replace the microbial lipases currently available on the market.
  • a nucleotide sequence encoding for SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 was integrated into a targeted locus of an expression host such as Pichia pastoris.
  • Codon-optimized genes encoding for SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 may or not be used.
  • the nucleotide sequence encoding for SEQ ID NO: 1 is, for example, SEQ ID NO: 12 or SEQ ID NO: 23.
  • the nucleotide sequence encoding for SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 can be, for example, SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 18 or SEQ ID NO: 19 or SEQ ID NO: 20, or SEQ ID NO: 21 or SEQ ID NO: 22, respectively.
  • Pichia pastoris may be replaced by Saccharomyces spp., Saccharomyces cerevisiae, Schizosaccharomyces spp., Candida spp., Candida cylindracea, Kluyveromyces spp., Fusarium spp., Fusarium oxysporium, Aspergillus spp., Aspergillus oryzae or Aspergillus niger, Trichoderma spp., Escherichia coli or Bacillus spp., Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, Streptomyces spp., Streptomyces l
  • a nucleotide sequence encoding for SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 may also be cloned into a conventional cloning and expression vector.
  • Codon-optimized genes encoding for SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 may or not be used.
  • the nucleotide sequence encoding for SEQ ID NO: 1 may be, for example, SEQ ID NO: 12 or SEQ ID NO: 23.
  • the nucleotide sequence encoding for SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 can be, for example, SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 18 or SEQ ID NO: 19 or SEQ ID NO: 20, or SEQ ID NO: 21 or SEQ ID NO: 22, respectively.
  • the recombinant cloning and expression vector may be further introduced in a host cell, such as a recombinant host cell, using standard transformation or transfection protocols known to the person skilled in the art and explained in literature (for example, in J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory manual, 2 nd edition, books 1- 3, Cold Spring Harbor Laboratory Press).
  • the microbial lipases (SEQ ID NO: 1-11) herein disclosed were independently expressed as follows. Pichia pastoris was used as a host for lipase expression. Pichia pastoris was grown on YPD agar plates (Invitrogen) under selective conditions (100 pg/mL Zeocin) at 30°C for 16h. Seed fermentation was performed by inoculating 3 mL YPD liquid medium (Invitrogen) including 100 pg/mL Zeocin with a single colony from the respective YPD agar plates, followed by incubation at 30°C and 250 rpm agitation for 16h.
  • Main fermentation was performed in 4 mL total volume by diluting seed fermentations with BMGY medium (Invitrogen) including 100 pg/mL Zeocin to an O ⁇ boo of 0.2, followed by incubation at 30°C and 225 rpm agitation for 3 days in 24-well culture plates.
  • Main fermentation samples were centrifuged to precipitate expression host cells and the supernatant was sterile filtered at 0.2 pm.
  • the filtrate was used directly for lipase enzymatic activity characterization without further treatment.
  • the activity was determined in tri butyrin/agarose emulsion, for example as described below for Example 2 or by IDF method. In particular, the IDF method was used for dosing in Examples 3-6.
  • Enzymatic activity for short chain fatty acids in plate a tributyrin/agarose emulsion was generated by dissolving 1.4% low-melting agarose in 250 mM sodium acetate buffer pH 5.25 including 40 mM CaCh, addition of 1% (v/v) tributyrin to the solubilized agarose at 60°C, and subsequent emulsification for 2 min using an IKA T25 Ultra-Turrax emulsifier at speed 13.5. A total volume of 50 pL emulsion was transferred to each well of a transparent 96-well assay plate and allowed to solidify at room temperature.
  • Lipase reaction was started by adding 20 pL enzyme on top of the emulsion layer, in particular wherein 20 pL enzyme correspond to 20 pL of filtrate obtained from Example 1. Clearance of turbidity of the emulsion by enzymatic hydrolysis of substrate tributyrin was followed by measuring absorbance at 600 nm in a plate reader. Each assay was calibrated with various dilutions of commercial microbial lipase A with known enzymatic activity.
  • Enzymatic activity for long chain fatty acids in plate an olive oil/agarose emulsion was generated by dissolving 1.4% low-melting agarose in 250 mM sodium acetate buffer pH 5.25 including 40 mM CaCh, addition of 2.5% (v/v) olive oil and 0.001% rhodamine B (w/v) to the solubilized agarose at 60°C, and subsequent emulsification for 2 min using an IKA T25 Ultra- Turrax emulsifier at speed 13.5. A total volume of 150 pL emulsion was transferred to each well of a non-transparent black 96-well assay plate and allowed to solidify at room temperature.
  • Lipase reaction was started by adding 30 pL enzyme on top of the emulsion layer, in particular wherein 30 pL enzyme correspond to 30 pL of filtrate obtained from Example 1. Binding of rhodamine B to long chain fatty acids released by enzymatic hydrolysis of substrate olive oil was followed by measuring fluorescence at 355 nm excitation and 590 nm emission in a plate reader. Each assay was calibrated with various dilutions of commercial microbial lipase A with known enzymatic activity.
  • Short chain fatty acid specificity C4/C16-C18 was determined, for each microbial lipase herein disclosed, by calculating the ratio of hydrolytic activities on substrate tributyrin (C4) and olive oil (C16-C18).
  • the ratio C /Ci 6 -Ci 8 of each lipase (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and/or SEQ ID NO: 11) is normalized for the ratio C4/C16-C18 of the microbial commercial lipase (A) used.
  • Table 1 shows that the microbial lipases herein disclosed (SEQ ID NO: 1-11) have a higher specificity for the short chain fatty acid butyric acid (C4) as compared to commercial microbial lipase A, in particular the microbial lipases of herein disclosed (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and/or SEQ ID NO: 11) are 2-770 times more C ⁇ fatty acid specific than the commercial microbial lipase A.
  • Short chain fatty acid specificity determined with TAG substrates tributyrin and olive oil of lipases with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and commercial microbial lipase A and respective improvement factor (IF4).
  • EXAMPLE 3 ENZYMATIC ACTIVITY IN CREAM
  • the lipases herein disclosed (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11), were additionally analyzed for their enzymatic activity towards milk fat and the released free fatty acids were quantified (Table 2).
  • the same procedure was conducted for a commercial microbial lipase (commercial microbial lipase A) from Rhizomucour miehei (Palatase®) available from Novozymes A/S.
  • Lipase activity on milk fat in cream was carried out as follows. Fresh whipping cream (38% fat) and equal amounts of water were mixed by stirring for 30 min. Enzymatic reaction was started by adding 20 pL lipase (from a 4000 LFU/L stock solution) to 1 mL cream/water mix and proceeded at 37°C for 20 hours, in particular this dosage of enzyme applies both to the commercial microbial lipase A and to lipases of SEQ ID NO: 1-11. Subsequently, free fatty acids were extracted from the reaction and quantified by gas chromatography-mass spectrometry (GC-MS), using standard protocols in the art for quantifying fatty acids. Alternatively the disclosure of Jong C., de and Badings H.T. in J. High Resolution Chromatography. 13, 84-98 (1990) can also be used.
  • GC-MS gas chromatography-mass spectrometry
  • Improvement factors IF4 and IF6 were calculated by dividing specificity values for C4 : o and C 6: o by the respective values of commercial lipase A.
  • Medium chain fatty acid specificities (Cs/ Ci 6 -Cis; Cio/ Ci 6 -Cis) were obtained by calculating the ratio of the molar fraction of Cs:o or Cio:o and the sum of molar fractions of Ci 6: o, Cis:o, Cis:i and Cis:2.
  • Improvement factors IF8 and IF10 were calculated by dividing specificity values for Cs : o and Cio : o, respectively, by the respective values of commercial lipase A.
  • the ratio C4/C16-C18 of each lipolytic lipase is determined by diving C4:o by the sum of Ci6:o, Ci8:o, C is : 1 and Ci8:2 of the respective lipolytic lipase.
  • the ratio C4/C16-C18 is indicative of the specificity of the respective lipase for the release of butyric acid (C4) from milk fat, relative to the release of fatty acids with chain lengths Ci 6 - Cis-
  • the ratio C6/C16-C18 of each lipolytic lipase is determined by diving C6:o by the sum of Ci6:o, Ci8:o, C is : 1 and Ci8:2 of the respective lipolytic lipase.
  • the ratio C 6 /Ci 6 -Cis is indicative of the specificity of the respective lipase for the release of caproic acid ⁇ Ce) from milk fat, relative to the release of fatty acids with chain lengths Ci 6 - Cis-
  • the ratio Cs/Ci 6 -Cis of each lipolytic lipase is determined by diving Cs:o by the sum of Ci 6: o, Ci8:o, C is : 1 and Ci8:2 of the respective lipolytic lipase.
  • the ratio Cs/Ci 6 -Cis is indicative of the specificity of the respective lipase for the release of caproic acid (Cs) from milk fat, relative to the release of fatty acids with chain lengths Ci 6 - Cis-
  • the ratio C10/C16-C18 of each lipolytic lipase is determined by diving Cio:o by the sum of Ci6:o, Ci 8: o, C is : 1 and Ci8:2 of the respective lipolytic lipase.
  • the ratio C10/C16-C18 is indicative of the specificity of the respective lipase for the release of caproic acid (Cio) from milk fat, relative to the release of fatty acids with chain lengths Ci 6 - Cis-
  • Short chain and medium chain fatty acid specificities C 4 /C 16 -C 18 , Ce/Cie-Cis, Cs/Ci 6 - Ci 8 , C 10 /C 16 -C 18
  • improvement factors IF4, IF6, IF8, IF10
  • SEQ ID NO: 1 SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.
  • All the microbial lipolytic enzymes herein disclosed show an IF4 higher than 1, in cream, meaning they have a higher specificity for the short chain fatty acid butyric acid (C4 : o) compared to commercial lipase A (Table 3) and can, therefore, be successfully used in a process for producing a food product wherein the presence of short chain fatty acid butyric acid (C 4: o) is needed and desired, such as for reducing soapiness and increase in butyric flavors in a food product.
  • Microbial lipolytic enzymes with SEQ ID NO: 1-7 and SEQ ID NO: 9-11 show an IF6 higher, than 1 in cream, meaning they have a higher specificity for the short chain fatty acid caproic acid (C 6: o) compared to commercial lipase A.
  • Microbial lipolytic enzymes with SEQ ID NO: 1-6 and SEQ ID NO: 8-11 show an IF8 higher, than 1 in cream, meaning they have a higher specificity for the medium chain fatty acid caprylic acid (Cs:o) compared to commercial lipase A .
  • Cs:o medium chain fatty acid caprylic acid
  • Microbial lipolytic enzymes with SEQ ID NO: 1-3, SEQ ID NO: 6-8 and SEQ ID NO: 10-11 show an IF10 higher, than 1 in cream, meaning they have a higher specificity for the medium chain fatty acid capric acid (Cio : o) compared to commercial lipase A.
  • Microbial lipolytic enzyme with SEQ ID NO: 1 has a higher specificity for C 4: o than for any other fatty acid, in cream. The same applies for microbial lipolytic enzymes having SEQ ID NO: 3 or SEQ ID NO: 9 (Table 2).
  • SEQ ID NO: 1 was further tested in cheese, in particular in Feta cheese and in Provolone cheese (Table 4).
  • Example 4 was carried out with an identical dosage of enzyme (commercial microbial lipase A and SEQ ID NO: 1), in particular with a dosage of about 1.7 LFU/L of enzyme per liter of milk was used for the production of Feta cheese and between 5-10x for the Provolone cheese. The dosage was determined by the IDF method.
  • Microbial lipase SEQ ID NO: 1 was added during the cheese making process.
  • the cheese making process of, for example Feta cheese or Provolone cheese, is well known in the art and to the skilled person.
  • free fatty acids were extracted from cheeses after 1 month of ripening and quantified by GC-MS or as disclosed in Example 3. Obtained fatty acid quantities of lipase reactions were corrected for the respective quantities determined for a similar cheese without added lipase.
  • Short chain fatty acid specificities were obtained by calculating the ratio of the molar fraction of C 4: o or C 6: o and the sum of molar fractions of Ci 6: o, Ci 8: o, Ci 8:i and Ci 8:2 - Improvement factors IF4 and IF6 were calculated by dividing specificity values for C 4: o and Oe : o by the respective values of commercial lipase A. Table 4.
  • SEQ ID NO: 1 has a higher specificity for C 4: o-fatty acids than commercial microbial lipase A, in Feta cheese and in Provolone cheese, and a higher specificity for C 4: o-fatty acids than for any other fatty acid, both Feta cheese and Provolone cheese, which is in line with the results obtained in cream (Example 3). Further, SEQ ID NO: 1 also has a higher specificity for C6 : o- fatty acids than commercial microbial lipase A, in Feta cheese and in Provolone cheese, which is also in line with the results obtained in cream (Example 3).
  • SEQ ID NO: 1 also shows a lower specificity for medium to long chain fatty acids such as Cs : o, Cio:o,Ci4:o,Ci6:o,Ci8:o,Ci8:i or Ci8:2 fatty acids when compared to commercial microbial lipase A in Feta cheese. Furthermore, SEQ ID NO: 1 also shows a lower specificity for medium to long chain fatty acids such as Cs:o,Cio:o,Ci2:o,Ci4:o,Ci6:o,Ci8:o,Ci8:i or Ci8:2 fatty acids when compared to commercial microbial lipase A in Provolone cheese.
  • a cheese produced using a microbial lipase comprising SEQ ID NO: 1 or wherein the microbial lipase is microbial lipase of SEQ ID NO: 1 is less soapy and more butyric than a cheese produced with a known microbial lipase.
  • Identical results are expected for SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and/or SEQ ID NO: 11.
  • microbial lipases herein disclosed are responsible for an increase in the C ⁇ fatty acid specificity in comparison with the prior art microbial lipases, thus leading to a reduced soapiness of cheese and an increase in the butyric flavor of cheese.
  • EXAMPLE 5 SENSORIAL EVALUATION
  • the Feta cheese was produced using identical conditions using a commercial A or SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5.
  • the amount or dosage of enzyme (commercial microbial lipase A, SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5) used was 1.7 LFU/L of milk determined by the IDF method (Table 5).
  • Table 5 shows a reduction of soapiness in Feta cheese, aged 3-months when SEQ ID NO: 1 or 3 or 5 is used when compared with commercial lipase A, while simultaneously an increase in the butyric flavor is observed for the same samples.
  • SEQ ID NO: 1, 2, 3, 6, 8, 10 and 11 were further tested in cheese, in particular in Feta cheese.
  • Example 6 was carried out with an identical dosage of enzyme (the commercial microbial lipase A and SEQ ID NO: 1, 2, 3, 6, 8, 10 and 11), in particular the a dosage of enzyme was of about 2.3 LFU/L of milk, determined by the IDF method, for the production of Feta cheese.
  • Microbial lipases SEQ ID NO: 1, 2, 3, 6, 8, 10 and 11 were individually added during the cheese making process. Subsequently, free fatty acids were extracted from cheeses after 1- month (Table 6) and 2-months (Table 8) of ripening and quantified by GC-MS or as disclosed in Example 3. Obtained fatty acid quantities of lipase reactions were corrected for the respective quantities determined for a similar cheese without added lipase.
  • Short- and medium-chain fatty acid specificities were obtained by calculating the ratio of the molar fraction of C4:o,C6:o,Cs:o or Cio:o and the sum of molar fractions of Ci6:o,Ci6:i,Ci8:o,Ci8:i, Ci8:2 and Cis:3 (Tables 7 and 9).
  • Improvement factors IF4, IF6, IF8 and IF10 were calculated by dividing specificity values for C4:o,Oe:oCs:o or Cio:o by the respective values of commercial lipase A (Tables 7 and 9).
  • the tested microbial lipolytic enzymes show an IF4 higher than 1, in Feta cheese, after 1- month of ripening.
  • the tested enzymes have a higher specificity for the short chain fatty acid butyric acid (C4 : o) compared to commercial lipase A (Table 7) and can, therefore, be successfully used in a process for producing a food product wherein the presence of short chain fatty acid butyric acid (C4 : o) is needed and desired, such as for reducing soapiness and increase in butyric flavors in a food product.
  • This observation, of an IF higher than 1 for the tested lipases is in line with the results of Example 3, where data was obtained in cream.
  • the tested microbial lipolytic enzymes show an IF4 higher than 1, in Feta cheese, after 2- month of ripening.
  • the tested enzymes have a higher specificity for the short chain fatty acid butyric acid (C4 : o) compared to commercial lipase A (Table 7) and can, therefore, be successfully used in a process for producing a food product wherein the presence of short chain fatty acid butyric acid (C4 : o) is needed and desired, such as for reducing soapiness and increase in butyric flavors in a food product.
  • Microbial lipolytic enzymes with SEQ ID NO: 1, 2, 3, 6, 8, 10 and 11 show an IF6 and IF8 higher than 1, meaning they have a higher specificity for the short chain fatty acid caproic acid (C 6: o) and for medium chain fatty acid caprylic acid (Cs : o) compared to commercial lipase A.
  • the tested enzymes with the exception of SEQ ID NO: 3, also shown an IF10 higher than 1, and have, therefore, a higher specificity for medium chain fatty capric acid (Cio : o) compared to commercial lipase A.
  • this invention relates to flavor enhancement but also with shortening of the ripening times for ripened cheeses.
  • the comparison between free fatty acid profiles after 1-month or 2-months of ripening shows that it is possible to successfully reduce ripening time from 2-months (or more) to 1-month (or less), while the cheese still presents the desired short-chain fatty acid profile.
  • Example 6 Identical results to those disclosed in Example 6 are also expected for other types of cheese, such as Pecorino, Parmesan, Grana Padano, Pgiano Reggiano, Romano, Chester, Danbo, Manchego, Saint Paulin, Cheddar, Monterey, Colby, Edam, Gouda, Muenster, Swiss type, Gruyere, Emmental; pasta filata cheeses such as Mozzarella, and Queso fresco cheese; fresh cheese such as Ricotta, Cream cheese, Neufchatel or Cottage cheese; cream cheese, white mold cheese such as Brie and Camembert cheese, blue mold cheese such as Gorgonzola and Danish blue cheese; processed cheese, cheese-like product, or an enzyme-modified cheese.
  • other types of cheese such as Pecorino, Parmesan, Grana Padano, Purgiano Reggiano, Romano, Chester, Danbo, Manchego, Saint Paulin, Cheddar, Monterey, Colby, Edam, Gouda, Muenster, Swiss type
  • the present invention discloses microbial wild-type lipases with a higher specificity for short chain fatty acids, specially C ⁇ fatty acid, than the known prior art microbial lipases, leading to a reduction in soapiness and an increase in butyric flavors of a food product such as cheese. Therefore, this invention provides microbial wild-type lipases which fulfill the vegetarian and/or kosher requirements, while simultaneously showing higher specificity for short chain fatty acids, specially C ⁇ fatty acid and lower specificity for medium to long chain fatty acids from milk fat and/or other fat, specially showing lower specificity for long chain fatty acids from milk fat and/or other fat, thereby avoiding an extremely unpleasant soapy taste in a food product, like cheese.
  • the present invention can be applied to other kinds of food products wherein the purpose is to reduce the unpleasant soapy taste of said food product.
  • the ripening time of the food product can be reduced from 2-months to 1- month, in particular in Feta cheese, while still maintaining the proper and desired short-chain fatty acid profile.
  • Lipase comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or
  • SEQ ID NO: 3 or SEQ ID NO: 11, even more preferably SEQ ID NO: 1.
  • Lipase according to the previous item wherein said lipase has a higher specificity towards the release of C ⁇ fatty acids from a dairy composition comprising milk fat and/or other fat if compared with the release of Cio-fatty acids.
  • Lipase according to any of the previous items, wherein said lipase is a microbial lipase, preferably an isolated microbial lipase, a recombinant microbial lipase or a synthetic microbial lipase.
  • Isolated DNA sequence or recombinant DNA sequence or synthetic DNA sequence encoding a lipase according to any of the preceding items preferably wherein the isolated DNA sequence or recombinant DNA sequence or synthetic DNA is selected from a sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98% or at least 99%, or 100% sequence identity with SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 18 or SEQ ID NO: 19 or SEQ ID NO: 20 or SEQ ID NO: 21 or SEQ ID NO: 22 or SEQ ID NO: 23.
  • DNA sequence according to the preceding item further comprising a signal peptide sequence.
  • Vector comprising an isolated DNA sequence or recombinant DNA sequence or synthetic DNA sequence according to any of the previous items 7-8.
  • Host cell preferably a recombinant host cell, comprising a sequence according to any of the previous items 1-8 or a vector according to previous item 9.
  • Host cell according to the previous item wherein the host cell is selected from Pichia pastoris, Aspergillus or Bacillus subtilis, preferably Pichia pastoris or B. subtilis, more preferably Pichia pastoris.
  • Method for preparing a food product comprising a step of using a lipase comprising an amino acid sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11, preferably having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 9 or SEQ ID NO: 11; or using a DNA sequence encoding a lipase, wherein the DNA sequence is selected from a sequence having at least 90%, or at least 95%,
  • lipase is a microbial lipase, preferably an isolated microbial lipase, a recombinant microbial lipase or a synthetic microbial lipase. 17. Method according to any of the previous items 12-16, wherein the food product is a dairy product selected from a cheese, or a processed cheese, or a cheese-like product, or an enzyme-modified cheese, or a butter, or a yogurt, or a cream, or a seasoning.
  • the dairy product is a cheese, preferably wherein the cheese is Feta cheese, or Provolone cheese, or Pecorino cheese, or Parmesan cheese, or Grana Padano cheese, or Policyo Reggiano cheese, or Romano cheese, or Chester cheese, or Danbo cheese, or Manchego cheese, or Saint Paulin cheese, or Cheddar cheese, or Monterey cheese, or Colby cheese, or Edam cheese, or Gouda cheese, or Muenster cheese, or Swiss type cheese, or Gruyere cheese, or Emmental cheese; or pasta filata cheeses such as Mozzarella, and Queso fresco cheese; or fresh cheese such as Ricotta, Cream cheese, Neufchatel or Cottage cheese; or cream cheese, or white mold cheese such as Brie and Camembert cheese, or blue mold cheese such as Gorgonzola and Danish blue cheese.
  • the cheese is Feta cheese, or Provolone cheese, or Pecorino cheese, or Parmesan cheese, or Grana Padano cheese, or Policy Reggiano cheese, or
  • the food product is a non-dairy product, preferably a non-dairy cheese or vegan cheese, preferably wherein the lipase has a higher specificity towards the release of C ⁇ fatty acids from a composition comprising fat such as vegetable fat or from a water-in-oil emulsion if compared with the release of Cio-fatty acids or if compared with the release of Cs-fatty acids, or if compared with the release of C 6 -fatty acids, or if compared with the release of Ci2-fatty acids or if compared with the release of Cis : 2-fatty acids or if compared with the release of C M - fatty acids or if compared with the release of Cis : o-fatty acids or if compared with the release of Cis :i -fatty acids or if compared with the release of Ci 6 -fatty acids, or if compared with the release of Cs-fatty acids - Cis : 2-fatty acids, or if compared with
  • Food product comprising a lipase, wherein the lipase comprises an amino acid sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11, preferably having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 9 or SEQ ID NO: 11, and wherein the food product comprises more C ⁇ fatty acids than Cio-fatty acids or than any other fatty acid selected from C 6 -fatty acids or Cs-fatty acids or Ci2-
  • the food product is a dairy product selected from a cheese, a processed cheese, a cheese-like product, or a butter, a yogurt, a cream, a seasoning, preferably wherein the cheese is Feta cheese, or Provolone cheese, or Pecorino cheese, or Parmesan cheese, or Grana Padano cheese, or Policyo Reggiano cheese, or Romano cheese, or Chester cheese, or Danbo cheese, or Manchego cheese, or Saint Paulin cheese, or Cheddar cheese, or Monterey cheese, or Colby cheese, or Edam cheese, or Gouda cheese, or Muenster cheese, or Swiss type cheese, or Gruyere cheese, or Emmental cheese; or pasta filata cheeses such as Mozzarella, and Queso fresco cheese; or fresh cheese such as Ricotta, Cream cheese, Neufchatel or Cottage cheese; or cream cheese, or white mold cheese such as Brie and Camembert cheese, or blue mold cheese such as Gorgonzola and Danish blue cheese
  • a lipase for producing a food product, wherein the food product is a dairy product selected from a cheese, or a processed cheese, or an enzyme-modified cheese (EMC), or a cheese-like product, or a butter, or a yogurt, or a cream, or a seasoning, preferably for producing cheese, such as Feta cheese, or Provolone cheese, or Pecorino cheese, or Parmesan cheese, or Grana Padano cheese, or Personalgiano Reggiano cheese, or Romano cheese, or Chester cheese, or Danbo cheese, or Manchego cheese, or Saint Paulin cheese, or Cheddar cheese, or Monterey cheese, or Colby cheese, or Edam cheese, or Gouda cheese, or Muenster cheese, or Swiss type cheese, or Gruyere cheese, or Emmental cheese; or pasta filata cheeses such as Mozzarella, and Queso fresco cheese; or fresh cheese such as Ricotta, Cream cheese, Neufchatel or Cottage cheese; or cream cheese, or white mold
  • a lipase for producing or releasing more C ⁇ fatty acids and/or C 6 -fatty acids from a dairy composition comprising milk fat and/or other fat, such as vegetable fat, than for producing or releasing more Cs-fatty acids, Cio-fatty acids, Ci2-fatty acids, Ci4-fatty acids, Ci 6 -fatty acids, Cis : o-fatty acids, Cis :i -fatty acids, or Cis : 2-fatty acids, or Cis : 3-fatty acids, wherein the lipase comprises an amino acid sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11, preferably having at least
  • a lipase for producing or releasing more C4-fatty acids and/or C 6 -fatty acids in a food product than for producing or releasing more Cs-fatty acids, Cio-fatty acids, C12- fatty acids, Ci4-fatty acids, Ci 6 -fatty acids, Cis : o-fatty acids, Cis :i -fatty acids, or Cis : 2- fatty acids, or Cis : 3-fatty acids in the food product, wherein the lipase comprises an amino acid sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11, preferably having at least 90%, or at least 95%
  • the food product is a dairy product selected from a cheese and wherein the cheese is Feta cheese, or Provolone cheese, or Pecorino cheese, or Parmesan cheese, or Grana Padano cheese, or Policyo Reggiano cheese, or Romano cheese, or Chester cheese, or Danbo cheese, or Manchego cheese, or Saint Paulin cheese, or Cheddar cheese, or Monterey cheese, or Colby cheese, or Edam cheese, or Gouda cheese, or Muenster cheese, or Swiss type cheese, or Gruyere cheese, or Emmental cheese; or pasta filata cheeses such as Mozzarella, and Queso fresco cheese; or fresh cheese such as Ricotta, Cream cheese, Neufchatel or Cottage cheese; or cream cheese, or white mold cheese such as Brie and Camembert cheese, or blue mold cheese such as Gorgonzola and Danish blue cheese, preferably Feta cheese or Provolone cheese.
  • the cheese is Feta cheese, or Provolone cheese, or Pecorino cheese, or Parmesan cheese,
  • Lipase consisting of an amino acid sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11, preferably having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 9 or SEQ ID NO: 11; wherein the lipase is expressed in Pichia pastoris and has a higher specificity towards the release of C4-fatty acids from a dairy composition comprising milk fat and/or other fat if compared with the release of C
  • EP1776455 SEQUENCE LISTING
  • VTTGDVTW TGW SLGGNDGTLTASVSAHNWYLVDIDACK

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Abstract

La présente invention concerne une enzyme lipolytique microbienne de type sauvage, non modifiée, ayant une spécificité supérieure pour des acides gras à chaîne courte que pour des acides gras à chaîne longue, en particulier des acides gras longs. La présente invention concerne en outre un procédé de préparation d'un produit alimentaire, et l'utilisation de l'enzyme lipolytique microbienne de type sauvage, non modifiée.
PCT/EP2021/054825 2020-02-28 2021-02-26 Lipases, compositions, procédés et utilisations de celles-ci WO2021170799A1 (fr)

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WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023031266A1 (fr) * 2021-09-01 2023-03-09 Chr. Hansen A/S Yarrowia lipolytica et son utilisation pour la production de lipases libérant spécifiquement des acides gras à chaîne courte
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

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